Description
General Description
Natural human C4a is prepared by cleavage of human C4 protein by human C1s. It is produced during activation of both the classical and lectin pathways of complement. C4a
is a member of the anaphylatoxin family of three proteins (C3a, C4a and C5a) produced by the activation of complement (Hugli, T.E. et al. (1981)). It is an unglycosylated polypeptide
containing 77 amino acids with a molecular mass of 8,759 daltons. Many of the biological functions of C4a are similar to those of C3a, but the specific activities are far below those of
C3a. C4a activity is so low, in fact, that it was initially thought to be inactive. These measured activities include inducing muscle contraction in the guinea pig ileum test
(spasmogenic activity), desensitization of muscle to C3a stimulation suggesting that the same receptor for both C3a and C4a is involved (tachyphylactic activity) and inducing vascular
permeability in human skin (Gorski J.P. et al. (1979)). C4a does not show tachyphylactic activity against C5a or chemotactic activity. Removal of the C-terminal arginine by serum
carboxypeptidase N destroys all these activities (Meuller-Ortiz, S.L., et al. (2009)). C4a appears to act through the C3a receptor (C3aR) which is a G-protein coupled receptor found
widely distributed on peripheral tissues, lymphoid cells (neutrohphils, monocyes, and eosinophils) and in the central nervous system (astrocytes, neurons and glial cells) (Law,
S.K.A. and Reid, K.B.M. (1995)).
Physical Characteristics & Structure
Molecular weight: 8,759 calculated molecular mass. Observed mass (MALDI-TOF) is 8,762 + 9 mass units. pI = 9.0 to 9.5 (Gorski, J.P. et al. (1981))
Amino acid sequence (77 amino acids): NVNFQKAINE KLGQYASPTA KRCCQDGVTR LPMMRSCEQR AARVQQPDCR EPFLSCCQFA ESLRKKSRDK GQAGLQR
C4a is thought to be structurally very similar to C3a and C5a to which it is homologous. Thus its 3D structure is probably similar to the X-ray-derived crystal structure
of C3a (Huber, R. et al. (1980)) and the NMR derived structure of C3a: Nettesheim, D.G. et al. (1988); Murray, I. et al. (1999).
Function
See General Description above. C4a exhibits much weaker biological activities than C3a and C5a. Its activity in inducing erythema and edema in human skin is 25,000-fold
weaker than that of C5a and 100-fold weaker than C3a per nanomole. The spasmogenic activity of C4a is 2000-fold weaker than C5a and 100-fold weaker than that of C3a. Due to
these differences the role of C4a in these responses in vivo is thought to be negligible.
Assays
Two well established assays for C4a and C3a functional activities include induction of contraction in the guinea pig ileum and the permeation of a dye such as trypan blue from
the vasculature into skin. The anaphylatoxins also induce mast cell degranulation, (measured as histamine release), platelet aggregation, IL-1 release from monocytes and the release of
prostaglandins and leukotrienes from many cells and tissues. The other assays used for C3a (Dodds, A.W. and Sim, R.B. (1997)) should also respond to C4a, but few reports have
described utilizing these assays with C4a. ELISA kits for the assay of C4a levels (or more correctly C4a desArg levels) in blood and other fluids are sold by several companies.
These measurements are useful for detecting complement activation in vivo, but the interpretation of their meaning is complicated by the fact that clearance of the anaphylatoxins is rapid.
In vivo
Freshly drawn normal human serum contains significant levels of all three anaphylatoxins. Although these may represent the resting concentration in vivo it is difficult
to draw or store blood without some complement activation so a true in vivo concentration is difficult to determine. The presence of EDTA and Futhan in the collection tubes can
minimize this background (Pfeifer, P.H. et al. (1999)). Full activation of all C4 in blood (600 µg/mL) would result in ~3,400 nM C4a (~30 µg/mL). Due to the low biological activity of
C4a it could require activation of most of the C4 in a small region to achieve the micromolar C4a concentrations necessary to elicit a response.
Regulation
C4a levels are regulated by three processes: formation, inactivation and clearance. There are two enzymes that cleave C4 and release C4a: C1s and MASP-2. C4a is
“inactivated” by removal of its C-terminal arginine amino acid. The product C4a desArg (or C4a without the C-terminal arginine) is produced by the action of the plasma enzyme
carboxypeptidase N (Mueller-Ortiz S.L. et al. (2009)). The inactivation is rapid and most C4a is converted to C4a desArg within minutes of its formation. Inactivated C4a lack
measurable biological activity. Because of the large number of cells bearing C3a/C4a receptors (endothelial, immune, smooth muscle, neuronal, etc.) the capture, internalization
and digestion of C4a and C4a desArg probably results in its removal from circulation.